mRNA polyadenylation is a key processing event for almost all mRNAs in eukaryotic cells. It involves cleavage of maturing mRNAs at the 3'end and addition of a poly(A) tail. The poly(A) tail influences many aspects of mRNA metabolism, including mRNA stability, mRNA transport, and translation. Over half of all human genes have multiple polyadenylation sites, or poly(A) sites, leading to transcript variants containing distinct mRNA cis- regulatory elements and/or encoding protein isoforms. Alternative polyadenylation has been shown to be regulated in tissue- and condition-specific manners. A growing number of human diseases have been associated with altered polyadenylation activity. While the core elements for polyadenylation have been well characterized, little is known about the auxiliary elements that modulate polyadenylation activity. In particular, the role of RNA secondary structures in regulation of polyadenylation is completely unclear. The long term goal is to fully understand the mechanisms by which mRNA polyadenylation is regulated under different biological conditions. The specific aims of this study are 1) to systematically analyze different types of RNA structures associated with mammalian poly(A) sites by bioinformatics, and 2) to examine how RNA structures regulate mRNA polyadenylation by experimental assays. PUBLIC HEALTH RELEVANCE: mRNA polyadenylation is a key processing event for almost all mRNAs in eukaryotic cells. It involves cleavage of maturing mRNAs at the 3'end and addition of a poly(A) tail. The poly(A) tail influences many aspects of mRNA metabolism, including mRNA stability, mRNA transport, and translation. Over half of all human genes have multiple polyadenylation sites, or poly(A) sites, leading to transcript variants containing distinct mRNA cis- regulatory elements and/or encoding protein isoforms. Alternative polyadenylation has been shown to be regulated in tissue- and condition-specific manners. A growing number of human diseases have been associated with altered polyadenylation activity. While the core elements for polyadenylation have been well characterized, little is known about the auxiliary elements that modulate polyadenylation activity. In particular, the role of RNA secondary structures in regulation of polyadenylation is completely unclear. The long term goal is to fully understand the mechanisms by which mRNA polyadenylation is regulated under different biological conditions. The specific aims of this study are 1) to systematically analyze different types of RNA structures associated with mammalian poly(A) sites by bioinformatics, and 2) to examine how RNA structures regulate mRNA polyadenylation by experimental assays.